This proposal is concerned with a genetic and biochemical analysis of the regulation of initiation of replication of the antibiotic resistance plasmid R6K in Escherichia coli. This multi-copy plasmid is 38 kilobases in size and specifies resistance to the antibiotics ampicillin and streptomycin. Previous studies have defined a contiguous replication region that spans 4 kilobases in size. Within this region three origins of replication, designated Alpha, Beta and Gamma, have been identified and shown to function in vivo and in an in vitro replication system. In addition, a gene (pir) specifies a replication initiation protein (Pi) is located between the Gamma and Beta origins and is required for activity of all three origins. The Pi protein is multi-functional in that it exhibits positive and negative activity in the initiation of R6K replication and autoregulates the expression of the pir gene. The activity of the three replication origins also requires in cis seven 22 base pair direct repeats located in the Gamma-origin region. Molecular genetic and biochemical analysis will be directed at determining the role of the Pi protein in the regulation of initiation of plasmid R6K with emphasis on the nature of the interaction of the Pi protein with the direct repeat region, other segments of the R6K replicon, and E. coli replication proteins. The introduction of wild-type and mutant forms of the Pi protein and the direct repeats will be analyzed using gel electrophoresis, electron microscopy and NMR spectroscopy techniques. The effect of mutational changes in the direct repeat region and the Pi protein on the formation of RNA transcripts of the R6K replicons and replication also will be examined in vitro. An R6K gene product that directs the initiation of replication from the Beta-origin has been identified and will be characterized. In addition molecular genetic approaches will be directed at the mechanism of autoregulation of pir gene expression and the role of this autoregulation in plasmid copy number control. Finally, the role of E. coli host proteins in R6K replication will be explored by isolating and characterizing E. coli mutants that alter the replication properties of wild-type and mutant R6K plasmids. These experimental approaches are designed to elucidate the major components of the regulatory machinery and the nature of their interactions responsible for the control of the copy number of plasmid R6K, a member of a major group of plasmids characterized by the presence of direct repeats at a replication origin and a plasmid encoded replication protein.